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Abstract:

This invention relates to methods for quick and safe identification of
pathogens from biological samples. Iodinated resins may be employed to
destroy a pathogen while leaving the pathogen's DNA in a state that can
be analyzed. The DNA can then serve as a substrate for PCR analysis. The
use of these iodinated resins work in a significantly quicker manner than
prior art methods and allows scientists to spend a minimal time under
Biosafety Level Three (BSL-3) conditions.

Claims:

1. A method of analyzing a biological sample comprising the steps of: a)
providing a liquid sample containing a pathogen; b) contacting the liquid
sample with an iodinated resin; c) separating the iodinated resin from
the liquid; and d) determining the identity of the pathogen through
analyzation techniques.

2. The method of claim 1 in which the iodinated resin is a bead or
fragment.

3. The method of claim 1 in which the contacting of the sample is
enhanced with a Vortex apparatus.

4. The method of claim 3, wherein the separation of the liquid portion is
performed using a centrifuge.

5. The method of claim 1, wherein the analyzation technique is PCR.

6. A method of analyzing a biological sample comprising the steps of: a)
providing a liquid sample containing a pathogen; b) contacting the liquid
sample with an iodinated resin; c) centrifuging the liquid sample
containing the iodinated resin; d) removing a portion of the liquid
sample; e) adding the portion of the liquid sample to a PCR solution
specific for the pathogen; and f) quantifying the results of the PCR
process.

7. The method of claim 6, wherein the pathogen is a bacteria or a virus.

8. The method of claim 6, wherein the the contacting of the sample is
initiated with a Vortex apparatus.

9. A test kit for carrying out the process according to claim 1
comprising components to amplify nucleic acid.

10. A kit for analyzing a biological sample for a pathogen comprising a
iodinated resin in a sample holder which contains at least enough space
to add said biological sample.

11. The kit of claim 10, further comprising components to amplify nucleic
acid.

Description:

[0001] This invention relates to a novel broad spectrum rapid preparation
method for extracting DNA to be used for PCR amplification and other
molecular biological processes.

BACKGROUND OF THE INVENTION

[0002] The polymerase chain reaction (PCR) provides a method for
increasing the number of copies of a target sequence (amplifying the
signal) with or without having to culture the organism prior, thereby
allowing increased sensitivity in detecting DNA sequences present in
small amounts in samples with DNA from mixed populations (Ou, C. Y.; et.
al., Science 239:292-295; Saiki, R. K.; et. al. Science 239:487-494;
Saiki, R. K.; et. al. Science 230:1350-1354; Scharf, S. J.; et. al.
Science 233:1076-1078). The method involves melting the DNA and annealing
short oligomer primers to regions flanking a target sequence. DNA
polymerase is added to the mixture in the presence of free
deoxynucleotides, and DNA is extended from the primers across the target
region. The new duplexes are again melted and the process is repeated.
This results in the exponential accumulation of the specific target,
approximately 2n, where n is the number of cycles of melting and
primer extension. Single-copy genomic sequences can be amplified by a
factor of more than 10 million with high specificity. The method has been
refined by the use of a thermally stable polymerase isolated from Thermus
aquaticus (Taq), which obviates the need to add new polymerase after each
melting cycle, and has been shown to have great specificity.

[0003] An integral aspect of the fields of microbiology and the practice
of medicine is the ability to positively identify microorganisms at the
level of genus, species or serotype. Correct identification is not only
an essential tool in the laboratory but plays a significant role in the
control of microbial contamination in the processing of food stuffs,
production of agricultural products and monitoring of environmental media
such as ground water. Increasing stringency in regulations which apply to
microbial contamination have resulted in a corresponding increase in
industry resources which must be dedicated to contamination monitoring.

[0004] Owing to its ability to identify microorganisms and detect viral
DNA, PCR has become increasingly important in the detection and diagnosis
of diseases. Weisburg et al., (EP 517361), for example, discloses a
method for the detection and identification of pathogenic microorganisms
involving the PCR amplification of DNA of E. coli. Detection of bacterial
and viral DNA can significantly aid in the treatment of many diseases.
Accordingly, in today's medical field, it important for doctors and other
staff to quickly and accurately detect the presence of pathogens in the
biological or other fluids related to their patients.

[0005] Prior to performing a PCR, it is first necessary to purify the DNA
(or RNA) of a sample. Purification of DNA requires an extraction
procedure which generally involves disrupting the cellular membranes of
samples to be analyzed, denaturing proteins in the cells and separating
the DNA from the denatured protein and other cellular components. The DNA
extraction techniques available to clinicians and forensic scientists
have been time-consuming and laborious, often requiring multiple steps
and the use of hazardous reagents. Traditional DNA extraction techniques
include density gradient centrifugation, organic solvent precipitation
and/or salt precipitation. Besides the aforementioned problems, these
techniques increase the risk of cross-contamination from sample to
sample.

[0006] The problems associated with DNA extraction and purification have
resulted in newer methods designed to increase efficiency and safety.
Solid phase extraction methods such as those described in U.S. Pat. No.
5,234,809 and U.S. Pat. No. 7,238,530 have been applied to the
purification of nucleic acids including DNA and RNA. The method involves
lysing cells, binding the released DNA to a solid support, washing away
impurities and eluting the purified DNA. The preferred lysing agents are
the chaotropes guanidinium thiocyanate and guanidinium hydrochloride. The
solid support is preferentially a silica particle. While the method aims
at reducing the manipulation of the sample, it is still microorganism
specific and still requires multiple laboratory steps. Moreover, the DNA
extraction process requires the use of toxic reagents at high
concentrations.

[0007] Liquid phase methods have also been designed to simplify the DNA
extraction process. One such method relies on using the chelating resin
Chelex® 100, a styrene-divinylbenzene copolymer containing paired
iminodiacetate. This resin is capable of scavenging metal contaminants
(e.g. magnesium) that catalyze the degradation of nucleic acids.
Additionally, Chelex® 100 is able to disrupt cell membranes and
denature DNA. In practice, the sample is diluted in water in the presence
of proteinase K and the solution is incubated at 55° C. for
approximately one hour. The mixture is then heated to a temperature of
100° C. for another 15 minutes. The mixture is then shaken in a
vortex and centrifuged. Denatured protein and metal ions settle at the
bottom of the tube. An aliquot from the supernatant can be used to
perform the PCR. It is noted that the process requires multiple
manipulation, complex human interaction and heating to 100° C.,
which adds to processing time and increases processing hazards,
particularly when working with dangerous pathogenic material.
Furthermore, the process is not amenable to working with all types of
samples.

[0008] It is further noted that the majority of kits on the market provide
limited means of producing a sample that is non-pathogenic, forcing the
analyst to perform the PCR experiment under biohazard conditions. This
presents significant safety issues that have not been adequately
addressed in the art. As such, researches may need to perform experiments
in a Biosafety Level 3 or 4 (BSL-3 or BSL-4) laboratory, which further
adds to processing times.

[0009] Hence, there is exists a need to develop simplified, more rapid and
less hazardous procedures for analyzing biological samples. In
particular, there exists a need to develop a faster, safer and more
economical way to analyze pathogenic samples. Methods must be able to
deactivate the pathogen rapidly and efficaciously while retain the
integrity of the nucleic acid (e.g. DNA) to be amplified and analyzed.
Furthermore, a simplified procedure would allow non-expensive PCR
equipment to be used in small remote clinics and hospital centers to help
medical personnel to obtain a fast confirmed diagnostic in order to treat
their patients.

SUMMARY OF THE INVENTION

[0010] The present invention allows for a very rapid and simple (one-step)
method to lyse cells of a liquid sample (i.e. blood, saliva, urine and
others) in order to extract nucleic acids (e.g. DNA or RNA) from
pathogens found in the sample and simultaneously provides a biohazard
free sample for the laboratory personnel to handle without complex
equipment such as thermal or ultrasonic equipment. The DNA can serve as a
substrate for PCR or other microbiological procedures, which results in
the rapid amplification and identification of the pathogen, while at the
same time destroying the viability of the pathogen in question.

[0011] In one aspect of the present invention, an iodinated resin is used
as an active agent to lyse the cells of a liquid sample containing a
pathogen (e.g. virus or bacteria), and destroy the pathogenic capability
of the pathogen Nucleic acids from the lysed cells are then amplified and
identified using a PCR procedure. The iodinated resin does not negatively
impact the integrity of the DNA and hence, the performance and
sensitivity of the PCR are optimal.

[0012] One aspect of the current invention is a process for analyzing a
biological sample comprising the steps of: providing a liquid sample
containing a pathogen, contacting the liqiud sample with an iodinated
resin, separating the iodinated resin from the liquid and determining the
identity of the pathogen through analyzation techniques.

[0013] Yet another aspect of the present invention is a diagnostic kit for
analyzing a biological sample comprising the steps of: providing a liquid
sample containing a pathogen, contacting the liqiud sample with an
iodinated resin, separating the iodinated resin from the liquid and
determining the identity of the pathogen through analyzation techniques.

[0014] Yet another aspect of the present invention is a Pre-PCR diagnostic
kit comprising an iodinated resin, said kit allowing the elimination of
thermal and/or cooling and/or sonification steps and equipment.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention provides a rapid and safe method for
isolating the DNA of a pathogenic sample for use in PCR or other
microbiological procedures. The method involves using a demand
disinfectant iodinated resin as an active agent to lyse the cell membrane
of a bacterial or protective coat of a virus, thereby removing the
nucleic acid material from the pathogen. The iodinated resin destroys the
deleterious effects of a pathogen while preserving the DNA of said
pathogen. Surprisingly, it has been found that the new protocol
significantly cuts down, the number of laboratory steps and the time and
labor needed to analyze biological samples with PCR, while at the same
time destroying the pathogenic capabilities of the microbes in question.

[0016] An iodinated resin product has been proposed for use as a demand
disinfectant, namely a disinfectant wherein iodine is released almost
entirely on a demand-action basis. An iodinated resin (the "Triosyn"
resin), such as the one disclosed in U.S. Pat. No. 5,639,452 (the '452
patent) can be used to enhance the lysis and destroy the pathogenicity of
a microbe with no negative impact on the PCR process. The contents of the
'452 patent is incorporated by reference in its entirety herein. Triosyn
samples (beads, fragments, powders) all contain triiodide molecules,
which are the antimicrobial components being used to disrupt the
membranes of microorganisms such as viruses, bacteria and fungi. In
addition to the triiodide present, fragments and powders possess another
antimicrobial property which comes from their irregular shapes and their
sharp edges, thus allowing the Triosyn fragments or particles to
mechanically insert into the cell membrane of a bacteria or protective
coat of a virus.

[0017] Three such Triosyn resin powders used in accordance with the
present invention are referred to as Triosyn T-50 powder, Triosyn T-45
powder and Triosyn T-40 powder. The numbers refer to the approximate
weight percentage of iodine relative to the resin. Powders with other
weight percentages of iodine may also be used in accordance with the
present invention.

[0018] Different percentages of iodine in the iodinated resin powders will
confer different properties to the powder, in particular different levels
of lysis and biocidal activity. The particular resin used in the process
is based on the desired application. The amount of Triosyn resin beads in
a sample being processed for PCR will be in the range of about 0.0025
grams to about 0.5 grams per 500 μL of a bacterial suspension.

[0019] In one aspect of the present invention, a rapid and safe method for
purifying DNA or RNA from a liquid sample containing a pathogen is
provided. The sample can be, for example, blood, saliva, or urine. The
sample containing the pathogen is added to a sterile microtube. The
Triosyn beads or fragments are then added and the sample is vortexed.
Vortexing is done for a time between 1 minute and 5 minutes, depending on
the nature of the sample and iodinated resin active agent. The sample is
then centrifuged to allow the iodinated resin to migrate to the bottom of
the tube. Centrifuging can be done for approximately 1 to 10 minutes,
preferably about 5 minutes. Following centrifugation, the majority of the
DNA and the RNA is found in the supernatant rather than at the bottom of
the vessel. Likewise, impurities that would normally cause degradation of
the DNA migrate to the bottom of the tube. These impurities likely
include proteins which are denatured by the iodinated resin as well as
metals that are exchanged with the iodinated resin. Accordingly, the DNA
or RNA in the supernatant contains DNA or RNA that is viable and can be
used as a substrate for further applications including PCR. The iodinated
resin does not damage the DNA or RNA of the sample.

[0020] In an alternative embodiment, proteinase K may be added to the
sample containing the iodinated resin prior to vortexing. The proteinase
K will assist the iodinated resin in denaturing proteins that are in the
sample.

[0021] Once the sample is rendered harmless by the iodinated resin, the
biological sample may be safely examined by PCR analysis without further
preparation or process to determine which virus or bacteria is contained
in the sample. PCR amplifications are standard in the art. For example,
PCR amplification procedures have been described in U.S. Pat. Nos.
5,234,809, 5,928,906, and 7,238,530, all of which are hereby incorporated
by reference. Importantly, the iodinated resin does not have a negative
impact on the PCR process and enzymes. Thus, using the inventive
protocol, medical staff has the ability to quickly and safely determine
the nature of the virus or bacteria are present in the particular
biological sample. Moreover, owing to its simplicity and low cost, the
process enables PCR equipment to be located in small medical centers and
remote areas and allows the medical personnel to obtain rapid diagnostic
information. This is not available today since current protocols require
specific and complex diagnostic kits that necessitate the use of
expensive thermal and or sonification equipment to be able to execute the
test.

[0159] The following sections describe exemplary embodiments of the
present invention. It should be apparent to those skilled in the art that
the described embodiments of the present invention provided herein are
illustrative only and not limiting, having been presented by way of
example only. All features disclosed in this description may be replaced
by alternative features serving the same or similar purpose, unless
expressly stated otherwise. Therefore, numerous other embodiments of the
modifications thereof are contemplated as falling within the scope of the
present invention as defined herein and equivalents thereto.

Experimental Data of Activity of Iodinated Resins On Pathogens

[0160] The following examples show the ability of demand disinfectant
iodinated resins to eliminate the pathogenicity of microbes.

[0165] Triosyn T40 beads (0.025 grams) were added to a pre-labeled tube
into which 10 mL of a VRE suspension (one colony diluted with 300-500
μL) was added and the sample was then vortexed. The sample was
analyzed at time points of 0, 2, 5, 10, 15 and 30 minutes for the
presence of active VRE. It was found that at the 2 and 5 minute time
points there was a reduction of the active VRE and by the 10 minute time
point there was virtually no active VRE left in the tube. At the 15 and
30 minute time points there was no active VRE present. This experiment
was conducted with a control that vortexed the VRE suspension with no
Triosyn T40 beads present, resulting in no reduction in the amount of
active VRE.

Example 2

[0166] Triosyn T40 beads (0.025 grams) were added to a pre-labeled tube
into which 10 mL of a VRE suspension (one colony diluted with 300-500
μL) was added and the sample was then vortexed. The sample was
analyzed at time points of 0, 2, 5, 10, 15 and 30 minutes for the
presence of active VRE. It was found that at the 0 minute time point
there was a reduction of the active VRE and by the 2 minute time point
there was virtually no active VRE left in the tube. At the 5, 10, 15 and
30 minute time points there was no active VRE present. This experiment
was conducted with a control that vortexed the VRE suspension with no
Triosyn T50 beads present, resulting in no reduction in the amount of
active VRE.

Example 3

[0167] Triosyn T40 beads (0.025 grams) were added to a pre-labeled tube
into which 10 mL of a VRE suspension (one colony diluted with 300-500
μL) was added and the sample was then vortexed. The sample was
analyzed at time points of 0, 2, 5, 10, 15 and 30 minutes for the
presence of active VRE. It was found that at the 0 and 2 minute time
points there was a reduction of the active VRE and by the 5 minute time
point there was virtually no active VRE left in the tube. At the 10, 15
and 30 minute time points there was no active VRE present. This
experiment was conducted with a control that vortexed the VRE suspension
with no Triosyn T45 beads present, resulting in no reduction in the
amount of active VRE.

Example 4

[0168] Triosyn T40 beads (0.025 grams) were added to a pre-labeled tube
into which 10 mL of a VRE suspension (one colony diluted with 300-500
μL) was added and the sample was then vortexed. The sample was
analyzed at time points of 0, 2, 5, 10, 15 and 30 minutes for the
presence of active VRE. It was found that at the 0 minute time point
there was virtually no active VRE left in the tube, the fragments did not
need to vortexed. At the 2, 5, 10, 15 and 30 minute time points there was
no active VRE present. This experiment was conducted with a control that
vortexed the VRE suspension with no Triosyn T50 fragments present,
resulting in no reduction in the amount of active VRE.

Example 5

[0169] Purolite A-605 iodinated resin beads (0.025 grams) were added to a
pre-labeled tube into which 10 mL of a VRE suspension (one colony diluted
with 300-500 μL) was added and the sample was then vortexed. The
sample was analyzed at time points of 0, 2, 5, 10, 15 and 30 minutes for
the presence of active VRE. It was found that at the 0 minute time point
there was virtually no active VRE left in the tube, the fragments did not
need to vortexed. At the 2, 5, 10, 15 and 30 minute time points there was
no active VRE present. This experiment was conducted with a control that
vortexed the VRE suspension with no Purolite A-605 beads present,
resulting in no reduction in the amount of active VRE.

Example 6 (Control Example)

[0170] Triosyn 402-C1 resin beads (0.025 grams) were added to a
pre-labeled tube into which 10 mL of a VRE suspension (one colony diluted
with 300-500 μL) was added and the sample was then vortexed. The
sample was analyzed at time points of 0, 2, 5, 10, 15 and 30 minutes for
the presence of active VRE. It was found that the Triosyn 402-C1 resin
did not eliminate active VRE at any time point.

[0171] The results from Examples 1-5 show that the iodinated resin is
capable of completely destroying the pathogen's capability to infect a
host. Control resin beads (Example 6) without iodine were not capable of
deactivating the microbe. Based on these findings, we tested whether the
samples containing iodinated resin can serve as substrates in DNA
amplifications.

Experimental Procedure For Performing PCR Experiments

[0172] The results below show that the method of the present invention can
be used to effectively perform PCR on the DNA of a pathogen. As discussed
above, because the virility of the pathogen is effectively destroyed this
method allows researchers to perform the PCR experiments without
contamination or the need to work under stringent biohazard conditions.

Example 7

Fortuitim mac farland 3

[0173] In a sterile microtube, add 500 μl of a dilution at 1:500 of
a suspension of mycobacterium Fortuitum mac farland 3. [0174] Add 4 doses
(approximately 0.025 grams) of T50 Triosyn iodinated resin beads. [0175]
Vortex for 2 minutes at maximum speed. [0176] Centrifuge for 5 minutes
and test 5 μl of the supernatant in PCR reaction mixture specific for
mycobacteria. Quantification of the results of the PCR indicated that the
DNA prepared by the procedure is of high quality which can be amplified
and analyzed successfully. Accordingly, the iodinated resin did not have
a detrimental impact on the DNA.

[0177] We suspected that the high quality of the PCR amplification was
partially related to the absence of viable microbes in the PCR solution,
which would negatively impact the PCR procedure. We tested the
microbiological efficiency of the Triosyn T50 beads by inoculating
Loewenstein Jensen tubes with the supernatant to verify the
presence/absence of bacterial development after 72 hours. The samples
prepared with iodinated resin beads showed no bacterial growth after 72
hours. Control samples without iodinated resin beads showed significant
bacterial growth after 72 hours (nbre of colony>200).

[0185] The results shown in Examples 7-10 show that the iodinated resin is
capable of completely destroying the pathogen's capability to infect a
host. Additionally, the methodology allows the user to perform high
quality PCR amplifications on the samples without possible infection from
the pathogen. Control experiments with an Amberlite resin (non-iodinated
resin) did not allow for any PCR amplification. Hence, the iodinated
resin does not destroy the integrity of the microbe's DNA. Moreover,
experiments can be performed without detergents, chemical additives,
filtration cooling, sonification and heating, thus greatly expediting the
process. Accordingly, the performance time of the PCR procedure is
significantly reduced.

Patent applications by Pierre J. Messier, Quebec CA

Patent applications in class Involving virus or bacteriophage

Patent applications in all subclasses Involving virus or bacteriophage